OBJECTIVE: To obtain information on the growth inhibition effect of arsenic compounds and gene expression profiles using cDNA microarray technique in SiHa cell lines. METHODS: We cultured 103 SiHa cell in 96 well plate and we investigated growth inhibition effects using MTT assay and also we performed gene expression profile experiment using 384 cDNA chip in SiHa cell after exposure of arsenics (As2O3, As4O6 - 1 (micro)M) for 48 hrs. RESULTS: Arsenics (As2O3, As4O6) inhibit the growth of SiHa cells (As2O3: 0.5, 1, 2, 3, 4, 5 (micro)M - 9.2, 56, 89, 93, 96, 96%, As4O6: 0.5, 1, 2, 3, 4, 5 (micro)M- 54, 84, 84, 85, 85, 87%) in 4 days culture. As2O3 and As4O6 induced apoptosis in SiHa cells. After exposure of As2O3, 47 genes were changed more than 2 times (eg, thymidylate synthetase, cyclin B1, CDC 20). In case of As4O6, 78 genes were changed more than 2 times (eg, CDC 20, cyclin B1, primase, proliferating cell nuclear antigen). CONCLUSION: we observed arsenic compound (As2O3, As4O6) inhibit the growth of SiHa cell. In gene expression profiling experiment, 78 genes was changed the expression level 2 times more than that of reference RNA after treatment of As4O6 and 47 genes after treatment of As2O3. Through these result, we thought more study need in functional genomics after arsenic treated cervical cancer cells.
Apoptosis ; Arsenic* ; Arsenicals ; Cell Line ; Centers for Disease Control and Prevention (U.S.) ; Cyclin B1 ; DNA Primase ; DNA, Complementary* ; Gene Expression Profiling ; Gene Expression* ; Genomics ; Oligonucleotide Array Sequence Analysis* ; RNA ; Thymidylate Synthase ; Transcriptome* ; Uterine Cervical Neoplasms

During severe acute respiratory syndrome coronavirus (SARS-CoV) infection, the activity of the replication/transcription complexes (RTC) quickly peaks at 6 hours post infection (h.p.i) and then diminishes significantly in the late post-infection stages. This "down-up-down" regulation of RNA synthesis distinguishes different viral stages: primary translation, genome replication, and finally viron assembly. Regarding the nsp8 as the primase in RNA synthesis, we confirmed that the proteolysis product of the primase (nsp8) contains the globular domain (nsp8C), and indentified the resectioning site that is notably conserved in all the three groups of coronavirus. We subsequently crystallized the complex of SARS-CoV nsp8C and nsp7, and the 3-D structure of this domain revealed its capability to interfuse into the hexadecamer super-complex. This specific proteolysis may indicate one possible mechanism by which coronaviruses to switch from viral infection to genome replication and viral assembly stages.
Amino Acid Sequence ; Crystallography, X-Ray ; DNA Primase ; chemistry ; genetics ; physiology ; Humans ; Isoenzymes ; chemistry ; genetics ; physiology ; Molecular Sequence Data ; Protein Structure, Secondary ; RNA, Viral ; biosynthesis ; SARS Virus ; chemistry ; genetics ; physiology ; Sequence Alignment ; Severe Acute Respiratory Syndrome ; virology ; Virus Replication

DNA primase catalyzes de novo synthesis of a short RNA primer that is further extended by replicative DNA polymerases during initiation of DNA replication. The eukaryotic primase is a heterodimeric enzyme comprising a catalytic subunit Pri1 and a regulatory subunit Pri2. Pri2 is responsible for facilitating optimal RNA primer synthesis by Pri1 and mediating interaction between Pri1 and DNA polymerase α for transition from RNA synthesis to DNA elongation. All eukaryotic Pri2 proteins contain a conserved C-terminal iron-sulfur (Fe-S) cluster-binding domain that is critical for primase catalytic activity in vitro. Here we show that mutations at conserved cysteine ligands for the Pri2 Fe-S cluster markedly decrease the protein stability, thereby causing S phase arrest at the restrictive temperature. Furthermore, Pri2 cysteine mutants are defective in loading of the entire DNA pol α-primase complex onto early replication origins resulting in defective initiation. Importantly, assembly of the Fe-S cluster in Pri2 is impaired not only by mutations at the conserved cysteine ligands but also by increased oxidative stress in the sod1Δ mutant lacking the Cu/Zn superoxide dismutase. Together these findings highlight the critical role of Pri2's Fe-S cluster domain in replication initiation in vivo and suggest a molecular basis for how DNA replication can be influenced by changes in cellular redox state.
Amino Acid Sequence ; Cell Cycle ; Cell Proliferation ; Chromatin Immunoprecipitation ; Cysteine ; genetics ; metabolism ; DNA Primase ; genetics ; metabolism ; DNA Replication ; DNA, Fungal ; genetics ; DNA-Directed DNA Polymerase ; metabolism ; Immunoblotting ; Immunoprecipitation ; Iron ; metabolism ; Iron-Sulfur Proteins ; metabolism ; Molecular Sequence Data ; Mutation ; genetics ; Oxidative Stress ; Protein Binding ; Saccharomyces cerevisiae ; genetics ; growth & development ; metabolism ; Sequence Homology, Amino Acid ; Sulfur ; metabolism

AIMTo investigate apoptosis induced by 3,3'-diethyl-9-methylthia-carbocyanine iodide (DMTCCI), an inhibitor of DNA primase found in our previous study, and the mechanism of DMTCCI in human myelogenous leukemia HL-60 cells.

METHODSHL-60 cells were cultured in RPMI-1640 medium and treated with different concentrations of DMTCCI. MTT assay was used to detect growth inhibition. Flow cytometry and DNA ladders were used to detect apoptosis. Western blotting was used to observe the expression of survivin, Bcl-xL, Bad, Bax, Bcl-2, caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity was measured by ApoAlert Caspase-3 Assay Kit.

RESULTSDMTCCI inhibited proliferation of human leukemia HL-60 cells with IC50 value of 0.24 micromol x L(-1). The results of flow cytometry and DNA ladders showed that DMTCCI could induce apoptosis of HL-60 cells. The expression levels of protein survivin and Bcl-xL were down-regulated, Bad and Bax were up-regulated, while Bcl-2 protein had no change in response to DMTCCI treatment in HL-60 cells. Treatment of HL-60 cells with DMTCCI induced the proteolytic cleavage of caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity apparently increased at 3 h and reached a peak at 12 h after exposure to 1 micromol x L(-1) of DMTCCI in HL-60 cells.

CONCLUSIONDMTCCI inhibited proliferation and induced apoptosis of human leukemia HL-60 cells. Bcl-2 family proteins, survivin and caspases family proteins might play a role in the apoptosis process induced by DMTCCI.

Apoptosis ; drug effects ; Carbocyanines ; pharmacology ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; DNA Damage ; DNA Fragmentation ; drug effects ; DNA Primase ; antagonists & inhibitors ; Flow Cytometry ; HL-60 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Leukemia, Myeloid ; metabolism ; pathology ; Microtubule-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-Associated Death Protein ; metabolism ; bcl-X Protein ; metabolism